Although there are numerous methods for the quantitative determination of glucose in biological fluids, there is a need for a simple, rapid, highly sensitive, accurate and reproducible means which can be easily miniaturized, inexpensively produced, and which is inexpensive to use. Such means would be especially useful, convenient and less painful to the patient when screening for and monitoring diabetes in the human if only a few drops of blood were required for a reliable test. Such a simple to use, rapid and reliable means to assay glucose could be used to monitor blood glucose levels at the hospital bedside, in a physician's office and as a home diagnostic tool to monitor the glycemic state of diabetics. The usefulness of such means would be enhanced if it had sufficiently sensitivity and accuracy to be applied to the quantitative determination of glucose concentrations in urine, which generally are far lower than in the blood. In addition, a simple, rapid, economical and convenient means which can be applied to the on-site monitoring of glucose concentrations during food processing and in agricultural products is needed. Furthermore, there is a need to increase the convenience and rapidity of glucose assays and to reduce the possibility of inaccurate results due to operator error in the preparation and use of aqueous solutions, by eliminating the need to prepare or mix such solutions prior to performing the assay.
Amperometric assay is an approach to the rapid assay of glucose in human or animal blood and other biological fluids which has been tried with varying degrees of success. Such assays utilize sensing electrodes in conjunction with a single redox mediator and a combination of oxidative and hydrolytic enzymes.
For example, Zhao et al (Biosensors & Bioelectronics vol. 8, pp 483-491, 1993) use tetrathiafulvalene (TTF) as a mediator dissolved in silicone oil and embedded in a graphite disc electrode in combination with immobilized glucose oxidase. Matthews et al (The Lancet, pp. 778-779, 1987) describe a pen sized digital blood glucose meter that uses ferrocene as a mediator, and a single use disposable strip on which dry glucose oxidase is deposited. Earlier work by Cass et al (Analytical Chemistry, vol. 56, pp 667-661 (1984) demonstrated the use of a ferrocene mediated enzyme electrode for the amperometric determination of glucose. Persson et al (Biosensors & Bioelectronics, vol. 8, pp 81-88 1993) described amperometric biosensors based on redox polymer-mediated electron transfer from NADH to carbon paste electrodes. Atanasov et al (Biosensors & Bioelectronics vol. 7, pp 361-365 (1992) demonstrated amperometric glucose biosensors using a porous carbon black matrix in which either 1,1'-dimethylferrocene or nickelocene or tetracyanoquinodimethane served as mediators. Marcinkeviciene et al (Biosensors & Bioelectronics, vol. 8, pp 209-212 (1993) used a ferrocyanide mediator in a strip type glucose biosensor.
Although amperometric sensors using a single mediator can be used to assay glucose, none really provides the combination of a wide response range, rapid response, a high degree of accuracy and precision over a wide response range and high sensitivity so that even relatively low concentrations of glucose such as found in urine could be assayed. In addition, there is no glucose biosensor which combines these attributes in a manner that allows easy and convenient use by untrained consumers.